PROJECT SUMMARY Priming of T-cell responses by dendritic cells (DCs) is an essential initiation step for the adaptive immune response. This process requires intimate cell-cell interactions at a site termed the immunological synapse (IS). Mounting evidence from a variety of sources indicates that this process involves mechanotransduction. Although events on the T cell side of the IS have been extensively studied, little is known about the DC side of the interface, apart from the fact that an intact DC actin cytoskeleton is required. We have found that DC maturation involves changes in cytoskeletal protein expression associated with altered biophysical properties of the cell cortex. Using atomic force microscopy (AFM), we found that cortical stiffness increases from 2kPa to 3.5kPa upon LPS-induced maturation, a large change in biophysical terms. Our preliminary data suggest that changes in the expression or activation state of several key actin regulatory molecules are responsible for regulating DC stiffness. Remarkably, when T cells are stimulated on substrates of different compliance, they exhibit a sharp threshold for activation over the range observed during DC maturation. Thus, we hypothesize that cytoskeletal changes associated with DC maturation alter the stiffness of the DC cortex, and that these events represent a previously unappreciated mechanism through which T cell priming is regulated. To test this hypothesis, we will carry out two specific aims: First, we will characterize the cytoskeletal changes that modulate DC stiffness during maturation. On the basis of preliminary studies using pharmacological inhibitors, we will focus on WASp, formins, cofilin and ERM proteins. Using DCs from knockout mice or WT DCs expressing shRNA or dominant mutants, we will block the expression or activity of candidate proteins and test the effect on cortical stiffness and T cell priming (using peptide loading to focus analysis on events at the IS). In addition, we will manipulate DC cortical stiffness in ways that do not occur naturally, and ask whether this affects T cell priming. Second, we will test T cell priming on substrates that vary in stiffness over the physiological range we have defined in DCs. Using hydrogels coated with T cell ligands, we will determine which T cell subsets are stiffness sensitive, and how stiffness affects proliferation and effector lineage development. We will determine the contribution of TCR, CD28, and LFA-1 to stiffness sensing, and ask how actin dynamics at the IS respond to changes in stiffness. Finally, we will characterize stiffness effects on Ca2+ signaling and key phosphorylation events, with particular emphasis on molecules known to participate in mechanotransduction. If successful, this exploratory project will show that regulated changes in the biophysical properties of the DC cortex function as a previously undiscovered mechanism through which DCs tune the T cell response - a basic feature of DC maturation to be considered along with upregulation of costimulatory molecules and cytokines. Moreover, it will provide a molecular foundation for understanding how T cells sense DC stiffness, guiding future investigation of the underlying mechanobiology. !